ET is a toxic substance mainly formed of lipopolysaccharide existing in a cell wall outer membrane of gram-negative bacteria, and freed therefrom when the bacteria are killed. When ET is incorporated into a living body by contaminating of ET to an injectable pharmaceutical preparation or the like, ET causes adverse action such as fever and ET shock thereon. Moreover, ET can act also on a cell itself, and therefore has possibility of influencing an experiment using various kinds of cells. In order to prevent the adverse action of ET, research has been conducted on a method for removing ET from an injectable solution, a substance in contact with a bodily fluid, a laboratory reagent or the like.
As the method for removing ET, an adsorption process by activated carbon or an ion exchanger, a filtration process using a membrane, a membrane filter or the like, a decomposition process by high temperature and high pressure treatment, acid treatment or alkaline treatment have been known so far. In particular, as a method for adsorbing ET to remove the same, an adsorbent using as a ligand polymyxin being a basic antibiotic (see Non-patent literature No. 1, Patent literature No. 1, Patent literature No. 2, for example), and an adsorbent using as a ligand amikacin being a basic antibiotic or the like in a similar manner (see Patent literature No. 3, for example) or the like has been known. The adsorbents using the antibiotics as the ligands are known to adsorb ET, but simultaneously nucleic acid such as DNA also, and are unsuitable for purification of the nucleic acid. For example, in an experiment of removing ET, using polymyxin-agarose, from a DNA solution, a recovery ratio of DNA is 60% at a maximum, and more specifically, at least 40% of DNA is removed together with ET (see Non-patent literature No. 2, for example).
Research has also been conducted on a cationic (basic) adsorbent produced by introducing an amino group thereinto. For example, a cationic adsorbent produced by allowing diamine to react with methyl polyglutamate, and introducing an amino group thereinto is known (see Patent literature No. 4, for example). The adsorbent has a feature in which the adsorbent has a pore size of 1,000 or less in terms of molecular weight of dextran and is substantially non-porous, and thus can selectively remove ET under coexistence of albumin.
Research has also been conducted on an adsorbent using as a ligand a polymer cationic substance. As the polymer, polyethyleneimine, polyallylamine, polylysine or the like is used (see Non-patent literature No. 3 and Non-patent literature No. 4, for example). Among the absorbents, an adsorbent using epsilon polylysine as the ligand can remove ET from a protein solution particularly when exclusion limit molecular weight of a base material is 6,000 or less (see Patent literature No. 5, for example). The adsorbents using the basic (cationic) substances as the ligands can selectively remove ET from the protein solution, as in the case of the adsorbent in which epsilon polylysine is immobilized, but can selectively remove no ET from a solution of nucleic acid such as DNA. Meanwhile, report has been made on an adsorbent using as a ligand epsilon polylysine in which an amino group is partially modified with glycidyl ether or the like as the adsorbent for selectively removing ET from a solution in which highly acid substance such as heparin coexists (see Patent literature No. 6, for example). However, the ligand is basically cationic and heparin adsorption is reduced by modification, but cannot be always completely prevented.
Cyclodextrin (hereinafter, also referred to as “CD”) is widely known to be a molecule in which glucose is linked in a cyclic manner to include a compound that can be entered into an inside of a cycle. Report has been made on a method for producing CD polymer beads by crosslinking CD with epichlorohydrin (see Patent literature No. 7, Patent literature No. 8 and Patent literature No. 9, for example). Such a CD polymer or a polymer of CD and a polystyrene resin is known to be used as an adsorbent of an environmental hormone such as bisphenol A (see Patent literature No. 10 and Patent literature No. 11, for example). Moreover, such a CD polymer is known to be used for removal of chlorogenic acid from a coffee extract (see Patent literature No. 12, for example). However, no description has been made on use of the CD polymers for removal of ET. Moreover, all of the CD polymers are produced by using epichlorohydrin as a crosslinking agent. In Patent literature No. 8, a di-epoxy compound, diisocyanate, an acrylamide derivative and so forth are exemplified as the crosslinking agent in addition to epichlorohydrin, but no properties of CD polymer produced using a crosslinking agent material other than epichlorohydrin have been disclosed.
Meanwhile, an invention is disclosed in which CD is crosslinked with a diisocyanate compound to prepare a polymer, the polymer is mixed with an aqueous solution of alginic acid, and then the resulting mixture is added dropwise to an aqueous solution of calcium chloride to prepare a spherical CD polymer and a phenolic compound that influences an environment is adsorbed with the spherical polymer (see Patent literature No. 13, for example). However, no arts have been disclosed on adsorption capacity of the phenolic compound with the CD polymer alone before mixing with alginic acid or the like. Moreover, no description has been made on use of the CD polymer produced using the diisocyanate compound as the crosslinking agent for removal of ET.
Moreover, an attempt has been made on adsorption and removal of lipopolysaccharides in blood by using a polymer resin obtained by crosslinking CD with a crosslinking agent such as epichlorohydrin (see Patent literature No. 14, for example). However, no description has been made as to whether or not ET can be selectively adsorbed and removed using the CD polymer resin. Moreover, in Patent literature No. 14, isocyanate, polyamine, acrylate and carbonate are exemplified as the crosslinking agent in addition to epichlorohydrin, but no properties of the CD polymer resin produced using a crosslinking agent other than epichlorohydrin have been disclosed.
Moreover, an attempt has been made on selectively removing ET from a DNA solution using CD polymer beads obtained by crosslinking CD with epichlorohydrin (see Non-patent literature No. 5, for example). According to the CD polymer beads, adsorption of DNA is hardly caused, and ET can be selectively removed. However, ET that can be adsorbed and removed with the CD polymer beads is about 80% based on the total amount, and about 20% of ET is not removed and remains in the solution. More specifically, the CD polymer beads have had room for improvement in view of ET adsorption capacity.
Moreover, report has been made on a method for producing nucleic acid and/or oligonucleotide containing no or reduced ET for a gene therapy (see Patent literature No. 15). Patent literature No. 15 discloses a method for treating a nucleic acid solution subjected to ET removal treatment beforehand with a surfactant-containing ET removal buffer or with an alkali-containing buffer or SDS-containing buffer, or a nucleic acid solution subjected ET removal treatment by nickel chelate chromatography or chromatography using polymyxin, DNA ETOX or the like by using a support of inorganic chromatography or an ion exchanger for holding DNA with a high salt concentration to purify DNA. However, the method is significantly complicated and requires a large number of solvents and reagents such as salts, and much time.